The present invention relates generally to methods of treatment for renal disease. In particular, the invention relates to methods of treatment for conditions which place mammals, including humans, at risk of chronic renal failure. The methods involve the administration of certain morphogens, inducers of those morphogens, or agonists of the corresponding morphogen receptors, or implantation of renal cells induced with those morphogens.
The mammalian renal system serves primary roles both in the removal of catabolic waste products from the bloodstream and in the maintenance of fluid and electrolyte balances in the body. Renal failures are, therefore, life-threatening conditions in which the build-up of catabolites and other toxins, and/or the development of significant imbalances in electrolytes or fluids, may lead to the failure of other major organs systems and death. As a general matter, renal failure is classified as xe2x80x9cacutexe2x80x9d or xe2x80x9cchronic.xe2x80x9d As detailed below, the differences between these two conditions are not merely a matter of severity or rapidity but, rather, reflect differences in etiology, prognosis, and treatment.
Acute Renal Failure
Acute renal failure is defined as an abrupt cessation or substantial reduction of renal function and, in as many as 90-95% of cases, may be secondary to trauma, surgery or another acute medical condition. Acute renal failure may be due to pre-renal causes (e.g., decreased cardiac output, hypovolemia, altered vascular resistance) or to post-renal causes (e.g., obstructions or constrictions of the ureters, bladder or urethra) which do not directly involve the kidneys and which, if treated quickly, will not entail significant loss of nephrons or other damage to the kidneys. Alternatively, acute renal failure may be due to intrinsic renal causes which involve a more direct insult or injury to the kidneys, and which may entail permanent damage to the nephrons or other kidney structures. Intrinsic causes of acute renal failure include but are not limited to infectious diseases (e.g., various bacterial, viral or parasitic infections), inflammatory diseases (e.g., glomerulonephritis, systemic lupus erythematosus), ischemia (e.g., renal artery occlusion), toxic syndromes (e.g., heavy metal poisoning, side-effects of antimicrobial treatments or chemotherapy), and direct traumas.
The diagnosis and treatment of acute renal failure is as varied as its causes. In human patients, oliguria (urine output less than 400 ml/day) or anuria (urine output less than 50 ml/day) may be present in 50-70% of cases, BUN levels may climb 10-20 mg/dl/day or faster, plasma creatinine levels may climb 0.5-1.0 mg/dl/day, and metabolic acidosis is almost always present. If not treated, the electrolyte and fluid imbalances (e.g., hyperkalemia, acidosis edema) associated with acute renal failure may lead to life-threatening arrhythmia, congestive heart failure, or multiple organ system failures. Present therapies are typically directed at the underlying causes of the acute renal failure (e.g., pre-renal, post-renal, or infectious causes) and management of the complications. Due to the severity of acute renal failure, episodes rarely last longer than several weeks without mortality and are treated on an in-patient basis.
Chronic Renal Failure
Chronic renal failure may be defined as a progressive, permanent and significant reduction of the glomerular filtration rate (GFR) due to a significant and continuing loss of nephrons. Chronic renal failure typically begins from a point at which a chronic renal insufficiency (i.e., a permanent decrease in renal function of at least 50-60%) has resulted from some insult to the renal tissues which has caused a significant loss of nephron units. The initial insult may or may not have been associated with an episode of acute renal failure. Irrespective of the nature of the initial insult, chronic renal failure manifests a xe2x80x9cfinal common pathxe2x80x9d of signs and symptoms as nephrons are progressively lost and GFR progressively declines. This progressive deterioration in renal ftmction is slow, typically spanning many years or decades in human patients, but seemingly inevitable.
The early stage of chronic renal failure typically begins when GFR has been reduced to approximately one-third of normal (e.g., 30-40 ml/min for an average human adult). As a result of the significant nephron loss, and in an apparent xe2x80x9cattemptxe2x80x9d to maintain the overall GFR with fewer nephrons, the average single nephron GFR (SNGFR) is increased by adaptations of the remaining nephrons at both the structural and functional level. One structural manifestation of this adaptation, readily detectable by microscopic examination of biopsy samples, is a xe2x80x9ccompensatory hypertrophyxe2x80x9d of both the glomeruli and the tubules of the kidney, a process which literally increases the volume of filtrate which can be produced by each remaining nephron by literal enlargement of the glomeruli and tubules. Indeed, as a result of the hypertrophy or dilation of the collecting ducts, the urine of subjects with chronic renal failure often contains broad xe2x80x9ccasts,xe2x80x9d typically 2-6 times normal diameter, which aid in diagnosis and have also been referred to as xe2x80x9crenal failure casts.xe2x80x9d At the same time, there are functional changes in the remaining nephrons, such as decreased absorption or increased secretion of normally excreted solutes, which may be responses to hormonal or paracrine changes elsewhere in the body (e.g., increasing levels of parathyroid hormone (PTH) in response to changes in serum levels of calcium and phosphate).
These adaptations in early stage chronic renal failure are not successful in completely restoring GFR or other parameters of renal function and, in fact, subject the remaining nephrons to increased risk of loss. For example, the increased SNGFR is associated with mechanical stresses on the glomerulus due to hypertension and hyperperfusion. The loss of integrity of podocyte junctures leads to increased permeability of the glomerulus to macromolecules or xe2x80x9cleakinessxe2x80x9d of the glomerular capsule. Proliferative effects are also observed in mesangial, epithelial and endothelial cells, as well as increases in the deposition of collagen and other matrix proteins. Sclerosis of both the glomeruli and tubules is another common symptom of the hypertrophied nephrons and the risk of coagulation in the glomerulus is increased. In particular, these adaptations of the remaining nephrons, by pushing the SNGFR well beyond its normal level, actually decrease the capacity of the remaining nephrons to respond to acute changes in water, solute, or acid loads and, therefore, actually increase the probability of additional nephron loss.
As chronic renal failure progresses, and GFR continues to decline to less than 10% of normal (e.g., 5-10 ml/min), the subject enters end-stage renal disease (ESRD). During this phase, the inability of the remaining nephrons to adequately remove waste products from the blood, while retaining useful products and maintaining fluid and electrolyte balance, leads to a rapid decline in which many organ systems, and particularly the cardiovascular system, may begin to fail. For example, BUN and creatinine levels may be expected to rise and, at BUN levels of 60-100 mg/dl and serum creatinine levels of 8-12 mg/dl, a uremic syndrome will typically develop in which the kidneys can no longer remove the end products of nitrogen metabolism. At this point, renal failure will rapidly progress to death unless the subject receives renal replacement therapy (i.e., chronic hemodialysis, continuous peritoneal dialysis, or kidney transplantation).
Approximately 600 patients per million receive chronic dialysis each year in the United States, at an average cost approaching $60,000-$80,000 per patient per year. Of the new cases of end-stage renal disease each year, approximately 28-33% are due to diabetic nephropathy (or diabetic glomerulopathy or diabetic renal hypertrophy), 24-29% are due to hypertensive nephrosclerosis (or hypertensive glomerulosclerosis), and 15-22% are due to glomerulonephritis. The 5-year survival rate for all chronic dialysis patients is approximately 40%, but for patients over 65, the rate drops to approximately 20%.
Morphogens and Growth Factors
A great many proteins have now been identified which appear to act as morphogenetic or growth factors, regulating cell proliferation or differentiation. Typically these growth factors exert their effects on specific sets or subsets of cells or tissues. Thus, for example, epidermal growth factors, nerve growth factors, fibroblast growth factors, various hormones, and many other proteins inducing or inhibiting cell proliferation or differentiation have been identified and shown to affect some subgroup of cells or tissues.
One group of morphogenetic proteins, referred to herein as xe2x80x9cmorphogens,xe2x80x9d includes members of the family of bone morphogenetic proteins (BMPs) which were initially identified by their ability to induce ectopic, endochondral bone morphogenesis. Subsequent characterization of the nucleic acid and amino acid sequences of the BMPs has shown them to be a subgroup of the TGF-xcex2 superfamily of growth factors. Members of the morphogen family have now been shown to include the mammalian osteogenic protein-1 (OP-1, also known as BMP-7), osteogenic protein-2 (OP-2), osteogenic protein-3 (OP-3). BMP-2 (also known as BMP-2A or CBMP-2A), BMP-3, BMP-4 (also known as BMP-2B or CBMP-2B), BMP-5, BMP-6, Vgr-1, and GDF-1, as well as the Xenopus homologue Vgl and the Drosophila homologues DPP and 60A. Members of this family encode secreted polypeptides that share common structural features and that are similarly processed from a pro-protein to yield a carboxy terminal mature protein of approximately 110 amino acids. All members share a conserved pattern of cysteines in this domain and the active form of these proteins is either a disulfide-bonded homodimer of a single family member, or a heterodimer of two different members (see, e.g., Massague (1990) Annu. Rev. Cell Biol. 6:597; Sampath, et al. (1990) J. Biol. Chem. 265:13198).
The members of the morphogen family of proteins are expressed in a variety of tissues during development. BMP-3 for, example, has been shown to be expressed in developing human lung and kidney (Vukicevic et al. (1994) J. Histochem. Cytochem. 42:869-875), BMP-4 has been shown to be expressed in the developing limbs, heart, facial processes and condensed mesenchyme associated with early whisker follicles in embryonic mice (Jones, et al. (1991) Development 111:531-542), and OP-1 (BMP-7) has been shown immunohistochemically to be associated with basement membranes in human embryos, including those of the developing lungs, pancreas, skin, and convoluted tubules of kidneys (Vukicevic, et al. (1994) Biochem. Biophys. Res. Commun. 198:693-700). Some of the morphogens (e.g., OP-2 and BMP-2) were not detected in analyses of adult tissues, suggesting only an early developmental role for these morphogens (Ozkaynak, et al. (1992) J. Biol. Chem. 267:25220-25227). In contrast, high levels of murine OP-1 expression have been observed in adult mouse kidneys (Ozkaynak, et al. (1991) Biochem. Biophys. Res. Commun. 179:116-123). This suggests a possible role for OP-1 synthesized in the kidney as a paracrine regulator of bone growth, and would be consistent with the role of the kidneys in both calcium regulation and bone homeostasis.
A great variety of growth factors have been considered which may participate in the regulation of the growth and repair of renal tissues (reviewed in, e.g., Toback (1992) Kidney Intl. 41:226-246). For example, EGF, TGF-xcex1, TGF-xcex2, IGF-I, IGF-II, PDGF, FGF, Renin/Angiotensin II, IL-I and OP-I have all been found to be expressed by various adult renal cells or tissues and to have effects on renal cell proliferation or differentiation (see, Toback (1992) supra, Ozkaynak, et al. (1991) a). In addition, several of these have been found to be expressed in the developing kidney, including IGF-I, TGF-xcex2 and OP-1 (reviewed in, e.g., Bard, et al. (1994) Mech. Develop. 48:3-11).
Interestingly, TGF-xcex2 has been shown in a murine metanephric organ culture system to retard overall growth and segmental differentiation of all segments of developing nephrons except the thick ascending limb-early distal tubules (Avner and Sweeney (1990) Pediatr. Nephrol. 4:372-377). In addition, TGF-xcex2 expression has been found to be increased in several models of renal disease, suggesting that TGF-xcex2 mediated increases in the synthesis of extracellular matrix components may be involved in the etiology of diabetic nephropathy (or diabetic glomerulopathy or diabetic renal hypertrophy), renal fibrosis, glomerulosclerosis and glomerulonephritis, interstitial fibrosis. and hypertensive nephrosclerosis (Shankland, et al. (1994) Kidney Intl. 46:430-442; Yamamoto, et al. (1994) Kidney Intl. 45:916-927; Yamamoto, et al. (1993) PNAS 90:1814-1818; Tamaki, et al. (1994) Kidney Intl. 45:525-536; Border, et al. (1990) Nature 346:371-374; Hamaguchi, et al. (1995) Hypertension 26:199-207).
Also of interest is the fact that serum levels of human growth hormone (GH) are elevated in subjects with chronic renal failure (Wright et al. (1968) Lancet 2:798; Samaan and Freeman (1970) Metabolism 19:102). Recombinant GH has been shown to help maintain protein balance in malnourished chronic renal failure patients, and to promote xe2x80x9ccatch-upxe2x80x9d growth in children with chronic renal failure. It has been suggested that these effects are mediated by IGF-I (see, e.g., Kopple (1992) Miner. Electrolyte Metab. 18:269-275). Although some studies have found that the administration of IGF-I increases renal plasma flow and GFR in chronic renal failure patients (e.g., Guler, et al. (1989) PNAS 86:2868-2872; Hirschberg, et al. (1993) Kidney Intl. 43:387-397), other studies have found that this effect is merely transient (Miller, et al. (1994) Kidney Intl. 46:201-207).
Thus, although some growth factors have been shown to be expressed in both developing and adult renal tissues, and although at least one has been shown to increase renal function in the short term, none has yet been shown to be of therapeutic benefit in preventing, inhibiting, or delaying the progressive loss of renal function that characterizes chronic renal failure. A need remains, therefore, for treatments which will prevent the progressive loss of renal function which has caused almost two hundred thousand patients in the United States alone to become dependent upon chronic dialysis, and which results in the premature deaths of tens of thousands each year.
The present invention is directed to methods of treatment, and pharmaceutical preparations for use in the treatment, of mammalian subjects at risk of chronic renal failure, or at risk of the need for renal replacement therapy. Such subjects include subjects already afflicted with chronic renal failure, or which have already received renal replacement therapy, as well as any subject reasonably expected to suffer a progressive loss of renal function associated with progressive loss of functioning nephron units. Whether a particular subject is at riskis a determination which may routinely be made by one of ordinary skill in the relevant medical or veterinary art. Subjects at risk of chronic renal failure, or at risk of the need for renal replacement therapy, include but are not limited to the following: subjects which may be regarded as afflicted with chronic renal failure, end-stage renal disease, chronic diabetic nephropathy, hypertensive nephrosclerosis, chronic glomerulonephritis, hereditary nephritis, and/or renal dysplasia; subjects having a biopsy indicating glomerular hypertrophy, tubular hypertrophy, chronic glomerulosclerosis, and/or chronic tubulointerstitial sclerosis; subjects having an ultrasound, MRI, CAT scan, or other non-invasive examination indicating renal fibrosis; subjects having an unusual number of broad casts present in urinary sediment; subjects having a GFR which is chronically less than about 50%, and more particularly less than about 40%, 30% or 20%, of the expected GFR for the subject; human male subjects weighing at least about 50 kg and having a GFR which is chronically less than about 50 ml/min, and more particularly less than about 40 ml/min, 30 ml/min or 20 ml/min; human female subjects weighing at least about 40 kg and having a GFR which is chronically less than about 40 ml/min, and more particularly less than about 30 ml/min, 20 ml/min or 10 ml/min; subjects possessing a number of functional nephron units which is less than about 50%, and more particularly less than about 40%, 30% or 20%, of the number of functional nephron units possessed by a healthy but otherwise similar subject; subjects which have a single kidney; and subjects which are kidney transplant recipients.
The methods and compositions of this invention capitalize in part upon the discovery that certain proteins of eukaryotic origin, defined herein as morphogens, may be used in the treatment of subjects at risk, as defined herein, of chronic renal failure or the need for renal replacement therapy. Generally, the morphogens of the invention are dimeric proteins that induce morphogenesis of one or more eukaryotic (e.g., mammalian) cells, tissues or organs. Of particular interest herein are morphogens that induce morphogenesis at least of mammalian renal tissue, including formation of functional renal epithelium and, in particular, functional glomerular and tubular epithelium. Morphogens comprise a pair of polypeptides that, when folded, adopt a configuration sufficient for the resulting dimeric protein to elicit morphogenetic responses in cells and tissues displaying receptors specific for said morphogen. That is, morphogens generally induce all of the following biological functions in a morphogenically permissive environment: stimulating proliferation of progenitor cells; stimulating the differentiation of progenitor cells; stimulating the proliferation of differentiated cells; and supporting the growth and maintenance of differentiated cells. xe2x80x9cProgenitorxe2x80x9d cells are uncommitted cells that are competent to differentiate into one or more specific types of differentiated cells, depending on their genomic repertoire and the tissue specificity of the permissive environment in which morphogenesis is induced. Morphogens further can delay or mitigate the onset of senescence- or quiescence-associated loss of phenotype and/or tissue function. Morphogens still further can stimulate phenotypic expression of differentiated cells, including expression of metabolic and/or functional, e.g., secretory, properties thereof. In addition, morphogens can induce redifferentiation of committed cells under appropriate environmental conditions. As noted above, morphogens that induce proliferation and/or differentiation at least of mammalian renal tissue, and/or support the growth, maintenance and/or functional properties of mammalian nephrons, are of particular interest herein.
In preferred embodiments, the pair of morphogen polypeptides have amino acid sequences each comprising a sequence that shares a defined relationship with an amino acid sequence of a reference morphogen. Herein, preferred morphogen polypeptides share a defined relationship with a sequence present in morphogenically active human OP-1, SEQ ID NO: 4. However, any one or more of the naturally occurring or biosynthetic sequences disclosed herein similarly could be used as a reference sequence. Preferred morphogen polypeptides share a defined relationship with at least the C-terminal six cysteine domain of human OP-1, residues 43-139 of SEQ ID NO: 4. Preferably, morphogen polypeptides share a defined relationship with at least the C-terminal seven cysteine domain of human OP-1, residues 38-39 of SEQ ID NO: 4. That is, preferred morphogen polypeptides in a dimeric protein with morphogenic activity each comprise a sequence that corresponds to a reference sequence or is functionally equivalent thereto.
Functionally equivalent sequences include functionally equivalent arrangements of cysteine residues disposed within the reference sequence, including amino acid insertions or deletions which alter the linear arrangement of these cysteines, but do not materially impair their relationship in the folded structure of the dimeric morphogen protein, including their ability to form such intra- or inter-chain disulfide bonds as may be necessary for morphogenic activity. Functionally equivalent sequences further include those wherein one or more amino acid residues differs from the corresponding residue of a reference morphogen sequence, e.g., the C-terminal seven cysteine domain (also referred to herein as the conserved seven cysteine skeleton) of human OP-1, provided that this difference does not destroy morphogenic activity. Accordingly, conservative substitutions of corresponding amino acids in the reference sequence are preferred. Amino acid residues that are xe2x80x9cconservative substitutionsxe2x80x9d for corresponding residues in a reference sequence are those that are physically or functionally similar to the corresponding reference residues, e.g., that have similar size, shape, electric charge, chemical properties including the ability to form covalent or hydrogen bonds, or the like. Particularly preferred conservative substitutions are those fulfilling the criteria defined for an xe2x80x9caccepted point mutationxe2x80x9d in Dayhoff et al. (1978), 5 Atlas of Protein Sequence and Structure, Suppl. 3, ch. 22 (pp. 354-352), Natl. Biomed. Res. Found., Washington, D.C. 20007, the teachings of which are incorporated by reference herein.
In certain embodiments, a polypeptide suspected of being functionally equivalent to a reference morphogen polypeptide is aligned therewith using the method of Needleman, et al. (1970), J. Mol. Biol. 48:443-453, implemented conveniently by computer programs such as the Align program (DNAstar, Inc.). As noted above, internal gaps and amino acid insertions in the candidate sequence are ignored for purposes of calculating the defined relationship, conventionally expressed as a level of amino acid sequence homology or identity, between the candidate and reference sequences. xe2x80x9cAmino acid sequence homologyxe2x80x9d is understood herein to mean amino acid sequence similarity. Homologous sequences share identical or similar amino acid residues, where similar residues are conservative substitutions for, or xe2x80x9callowed point mutationsxe2x80x9d of, corresponding amino acid residues in an aligned reference sequence. Thus, a candidate polypeptide sequence that shares 70% amino acid homology y with a reference sequence is one in which any 70% of the aligned residues are either identical to or are conservative substitutions of the corresponding residues in a reference sequence.
Of particular interest herein are morphogens, which, when provided to the kidney of a mammal, induce or maintain the normal state of differentiation and growth of nephron units. Of still more particular interest herein are morphogens which, when administered to a mammal, prevent, inhibit or delay the development of compensatory hypertrophy, including glomerular hypertrophy and/or tubular hypertrophy. Such morphogens can be used to treat a mammal at risk of chronic renal failure by preventing, inhibiting or delaying the progressive loss of functional nephron units and the consequent progressive loss of renal function.
The present invention alternatively can be practiced with methods and compositions comprising a morphogen stimulating agent or morphogen inducer in lieu of a morphogen. A xe2x80x9cmorphogen inducerxe2x80x9d is a compound that stimulates in vivo production, e.g., expression, of a therapeutically effective concentration of an endogenous morphogen in the body of a mammal sufficient to regenerate or maintain renal tissue and/or to inhibit additional loss thereof. Such compounds are understood to include substances which, when administered to a mammal, act on cells of tissue(s) or organ(s) that normally are competent to produce and/or secrete a morphogen encoded within the genome of the mammal. and which cause the endogenous level of the morphogen in the mammal""s body to be altered. Endogenous or administered morphogens can act as endocrine, paracrine or autocrine factors. That is, endogenous morphogens can be synthesized by the cells in which morphogenetic responses are induced, by neighboring cells, or by cells of a distant tissue, in which circumstances the secreted endogenous morphogen is transported to the site of morphogenesis, e.g., by the individual""s bloodstream. In preferred embodiments, the agent stimulates expression and/or secretion of an endogenous morphogen so as to increase amounts thereof in renal tissues.
In still other embodiments, an agent which acts as an agonist of a morphogen receptor may be administered instead of the morphogen itself. An xe2x80x9cagonistxe2x80x9d of a receptor means a compound which binds to the receptor and for which such binding has a similar functional result as binding of the natural, endogenous ligand of the receptor. That is, the compound must, upon interaction with the receptor, produce the same or substantially similar transmembrane and/or intracellular effects as the endogenous ligand. Thus, an agonist of a morphogen receptor binds to the receptor and such binding has the same or a similar functional result as morphogen binding (e.g., induction of morphogenesis). The activity or potency of an agonist can be less than that of the natural ligand, in which case the agonist is said to be a xe2x80x9cpartial agonist,xe2x80x9d or it can be equal to or greater than that of the natural ligand, in which case it is said to be a xe2x80x9cfull agonist.xe2x80x9d Thus, for example, a small peptide or other molecule which can mimic the activity of a morphogen in binding to and activating the morphogen""s receptor may be employed as an equivalent of the morphogen. Preferably the agonist is a full agonist, but partial morphogen receptor agonists may also be advantageously employed. Methods of identifying such agonists are known in the art and include assays for compounds which induce morphogen-mediated responses (e.g., induction of differentiation of metanephric mesenchyme, induction of endochondral bone formation, and the like). Such an agent may also be referred to as a morphogen xe2x80x9cmimic,xe2x80x9d xe2x80x9cmimetic,xe2x80x9d or xe2x80x9canalog.xe2x80x9d
The morphogens, inducers and agonists of the invention may be administered by any route of administration which is compatible with the selected agent, and may be formulated with any pharmaceutically acceptable carrier appropriate to the route of administration. Preferred routes of administration are parenteral and, in particular, intravenous, intraperitoneal, and renal intracapsular. Treatments are also preferably conducted over an extended period on an outpatient basis with daily dosages for morphogens in the range of about 0.01-1000 xcexcg/kg body weight, and more preferably about 0.1-100 xcexcg/kg body weight.
Finally, in yet further embodiments, renal cells may be implanted into the kidney of a subject at risk of chronic renal failure, or at risk of needing renal replacement therapy, in order to serve as a source of morphogen and/or to provide a source of additional functional renal tissue. These cells may be renal mesenchymal progenitor cells, or renal mesenchymal progenitor cells which have been induced to undergo metanephric differentiation. The cells may be derived from a donor (e.g., a tissue-type matched donor, sibling, identical twin), may be derived from a tissue culture (e.g., undifferentiated renal mesenchyme culture, fetal renal tissue culture), or may be explanted from the subject and then be re-implanted after proliferation and/or differentiation. Preferably, the cells are induced to undergo metanephric differentiation by treatment with a morphogen (e.g., OP-1) either before or after implantation.
The treatments of the present invention are useful in preventing, inhibiting or delaying the progressive loss of functional nephron units, and the consequent progressive loss of renal function, which typify chronic renal failure. As such they are of great value in preventing or delaying the need for chronic dialysis or renal replacement therapy in subjects with chronic renal insufficiency, or reducing the necessary frequency of chronic renal dialysis in subjects with end-stage renal disease. As such, they are useful in prolonging the lives, and in maintaining the quality of life, of subjects at risk of, or already afflicted with, chronic renal failure.